The recognized function of the ubiquitously expressed Na-H exchanger, NHE1, is to regulate intracellular pH (pHi) through electroneutral exchange of extracellular Na+ and intracellular H+. The applicant's laboratory recently determined that NHE1 has a previously unrecognized, critical role in regulating the organization of the actin cytoskeleton. Integrin receptors and the GTPase RhoA act upstream of NHE1 to stimulate its activity. Activation of NHE1, in turn is obligatory for integrin- and RhoA- induced cytoskeletal organization, including the formation of actin stress fibers, the expression, recruitment and assembly of focal adhesion-associated proteins, and the rate of cell attachment and spreading on a fibronectin matrix. Additionally, it was determined that the C-terminal cytoplasmic domain of NHE1 directly binds members of the ERM family of actin-binding proteins and that NHE1 and ERM proteins co-localize at sites of focal contact. ERM proteins, like NHE1, play a critical role in cytoskeletal remodeling in response to RhoA, and together these findings suggest that NHE1 may regulate cytoskeletal remodeling by providing a structural link to tether actin to the plasma membrane. Whether this action also involves the established role of NHE1 in ion translocation remains undetermined. The overall objective of this proposal is to determine how NHE1 mediates integrin- and RhoA- induced cytoskeletal organization by investigating the hypothesis that NHE1 contributes a specific signal that acts on distinct cytoskeletal targets. Studies in Aim 1 will determine whether NHE1 regulates cytoskeletal dynamics through a structural association with ERM proteins by using chimeric NHEs that either disrupt or promote ERM binding. Studies in Aim 2 will determine the role of ion translocation in cytoskeletal organization by using NHE1 tagged with pH-sensitive fluorescent probes to visualize localized pH(i) gradients, and by determining whether a translocation-defective NHE1 can rescue impaired cytoskeletal remodeling in NHE-deficient cells. Studies in Aim 3 will investigate the target of NHE1 actions by focusing on the bundling of actin filaments and proteins that control filament bundling. The organization of the actin cytoskeleton plays an important role in regulating a number of normal cellular processes, including proliferation, morphogenesis and wound healing, and it contributes to pathological conditions such as tumor metastasis and hypertension. Determining how NHE1 regulates the actin cytoskeleton will contribute to our understanding of the functional importance of ion exchangers, the control of actin filament organization, and the role the cytoskeleton plays in cellular processes.